PCR技术检测空肠弯曲菌的研究进展

空肠弯曲菌(Campylobacter jejuni)是一种人畜共患病病原菌,可以使人和动物引发多种疾病。目前,检测C.jejuni采用的国标方法是传统的培养法,但C.jejuni培养条件苛刻,且培养法存在操作繁琐、特异性不强、费时等缺点。聚合酶链式反应(polymerase chain reaction,PCR)以其快速、准确、灵敏度高、特异性强的特点,现已广泛应用于C.jejuni的检测,并成为目前快速检测临床与食品中C.jejuni最常用的的方法。本文综述了近年来利用RCR技术,包括常规PCR、多重PCR、巢式PCR、实时荧光定量PCR、PCR-酶联免疫吸附法、最大几率数-PCR、PCR-限制性片段长度多态性、PCR-变性高效液相色谱、PCR-变性梯度凝胶电泳和磁捕获-荧光PCR方法检测C.jejuni的研究进展,并针对这些PCR技术的原理、检测效果、优点和缺点等方面进行了分析比较,为有效控制和预防该菌引起的疾病提供重要信息。     

Serotypes and typability of Campylobacter jejuni and Campylobacter coli isolated from poultry products

Campylobacter infection is one of the most common bacterial enteric pathogens. Campylobacter jejuni and Campylobacter coli infections are mostly food- and waterborne and especially poultry is often assumed to be an important source. The heat-stable serotyping system (the 'Penner' scheme) was used to study the serotype distribution of C. jejuni and C. coli isolated from different food products of poultry origin sampled from retail outlets in Denmark. A total of 156 isolates were serotyped, 85% of these were C. jejuni and 15% were C. coli. The most common C. jejuni serotypes were O:2 (30%), O:1,44 (12%) and the O:4-complex (8%). O:46 was the most frequent serotype among C. coli isolates. These serotypes are also common among Danish clinical isolates and isolates from broiler chickens and cattle. Differences in serotype distribution were seen for different kinds of poultry products. Isolates from chicken products covered a large selection of serotypes. In contrast, the majority of the isolates from other product groups (turkey, poussin, wild birds) were concentrated on 1-3 serotypes. Using the standard procedure for antigen preparation and serotyping, 25 of the 156 strains (16%) were nontypable. This rate of nontypable isolates is significantly higher than experienced for isolates from other sources than food products, i.e faecal samples from animals and humans. Subculturing and re-typing of the nontypable isolates improved the typability. After two, five and 10 subcultures 16, six and one isolate became typable, respectively. Only three isolates (2%) remained nontypable after 10 subcultures.     

Detection of Campylobacter jejuni and Campylobacter coli in foods by enrichment culture and polymerase chain reaction enzyme-linked immunosorbent assay

A polymerase chain reaction (PCR) assay based on a solution hybridization format with colorimetric end-point detection (PCR ELISA) was investigated for the specific detection of Campylobacter jejuni and Campylobacter coli in food samples following enrichment culture. One hundred fifteen samples of raw meat and offal (poultry, porcine, ovine, and bovine), raw shellfish, and artificially contaminated milk were enriched in blood-free Campylobacter Enrichment Broth for 48 h. Enrichment cultures were subcultured to Campylobacter blood-free selective agar plates, and presumptive isolates were identified by phenotypic methods. DNA was extracted from 1-ml aliquots of the enrichment cultures using a rapid extraction method, and the DNA was used as the template in a PCR ELISA. A comparison of the PCR ELISA with the enrichment culture and subculture to selective agar method showed that the results of 112 of the 115 samples tested were in agreement by both methods. Seventy-one of the various food samples were positive in the PCR ELISA, and 70 samples were positive by culture. The PCR ELISA had a sensitivity of 99% and a specificity of 96%, with a positive predictive value of 97% and a negative predictive value of 98%. The PCR ELISA is a rapid, sensitive, and specific method for the detection of C. jejuni and C. coli in foods following enrichment culture and significantly reduces the time required for their detection.     

Metronidazole resistance in Campylobacter jejuni from poultry meat

The occurrence of metronidazole resistance was investigated among Campylobacter jejuni in raw poultry meat collected from supermarkets. MICs were determined by the agar dilution procedure in the testing range of 3 to 60 microg/ml metronidazole. The MICs showed a bimodal distribution with a significant proportion of metronidazole-resistant isolates among C. jejuni from raw broiler and turkey meat. Metronidazole resistance occurred most frequently among turkey meat isolates (P < 0.005). This is the first report of foodborne bacteria carrying metronidazole resistance.     

Evaluation of a commercial automated ELISA and PCR-method for rapid detection and identification of Campylobacter jejuni and C. coli in poultry products

Two rapid methods for detection and identification of Campylobacter jejuni andCampylobacter coli in naturally contaminated poultry products were evaluated for their applicability in routine studies. These methods were a commercial automated enzyme-linked immunosorbent assay (ELISA) for detection of thermophilic campylobacters in food, and C. jejuni -specific Polymerase chain reaction (PCR) analysis, which included a sample preparation method, based on Buoyant Density Centrifugation (BDC). Both methods were applied to the samples (97 samples) after a 48h enrichment procedure. The automated ELISA proved to be an easily repeatable and reliable method for detection of thermophilic campylobacters in food. The BDC-PCR method, which detected C. jejuni directly from the enrichment broth, was also reliable but not as simple as the ELISA. The analysis time for the ELISA-based method, starting from enrichment, took a minimum 2·5 working days, while the BDC-PCR method required 3 working days. Our study with a limited number of samples suggest that both detection methods are useful for monitoring C. jejuni on chicken meat.     

Prevalence and characterization of Campylobacter jejuni isolated from pasture flock poultry

The growing interest in organic and natural foods warrants a greater need for information on the food safety of these products. In this study, samples were taken from 2 pasture flock farms (N = 178; feed, water, drag swabs, and insect traps), pasture flock retail carcasses (N = 48) and 1 pasture flock processing facility (N = 16) over a period of 8 mo. A total of 105 Campylobacter isolates were obtained from 53 (30%), 36 (75%), and 16 (100%) samples from the farms, retail carcasses, and processing facility, respectively. Of the 105 isolates collected, 65 were C. jejuni, 31 were C. coli, and 9 were other Campylobacter spp. Using PCR, the C. jejuni isolates were further analyzed for virulence genes involved in colonization and survival (flaA, flaC, cadF, dnaJ, racR, cbrR), invasion (virB11, ciaB, pldA), protection against harsh conditions (sodB, htrA, clpA), toxin production (cdtA, cdtB, cdtC), siderophore transport (ceuE), and ganglioside mimicry (wlaN). In addition, the short variable region of the flaA locus (flaA SVR) was sequenced to determine the genetic diversity of the C. jejuni isolates. The flaA SVR diversity indices increased along the farm to carcass continuum. PCR-based analysis indicated a low prevalence of 5 genes involved in colonization (dnaJ, ciaB, pldA, racR, virB11). The results of this survey indicate that the prevalence of Campylobacter on organic retail carcasses is similar to prevalence reports of Campylobacter on conventional retail carcasses. However, the genetic diversity of the flaA SVR genotypes increased along the farm to carcass continuum that contrasted with conventional poultry studies. PRACTICAL APPLICATION: Campylobacter jejuni is a leading cause of foodborne illness with poultry and poultry products being leading sources of infection. Free-range and pasture flock chickens are becoming more popular; however, there is an inherent biosecurity risk that can increase the prevalence of foodborne pathogens in these flocks. This study aimed to determine sources and characterize C. jejuni isolated from pasture flocks.     

Rapid detection of Campylobacter jejuni in chicken rinse water by melting-peak analysis of amplicons in real-time polymerase chain reaction

Five DNA extraction protocols for the detection of Campylobacter spp. by polymerase chain reaction (PCR) were compared. A method involving Triton X-100 produced template DNA of sufficient quality to allow the detection of Campylobacter jejuni at levels of 100 CFU/ml in pure culture. Primers were designed on the basis of the cadF gene sequence. With a SYBR Green I real-time PCR assay, these primers amplified only sequences present in C. jejuni to produce a product with a melting temperature of 81.5 degrees C. None of the strains of Campylobacter coli, Campylobacter lari, or Campylobacter fetus tested produced this product during the PCR assay. Other noncampylobacter species tested were shown not to possess the cadF sequence. The real-time PCR combined with a rapid, simple Triton X-100 DNA extraction protocol made it possible to detect < 10 CFU of C. jejuni per ml of chicken rinse within 14 h.     

Incidence of Salmonella, Campylobacter jejuni, Campylobacter coli, and Listeria monocytogenes in poultry carcasses and different types of poultry products for sale on the Belgian retail market.

From January 1997 to May 1998, 772 samples of poultry carcasses and poultry products for sale on the retail market in Belgium were analyzed for the presence of Salmonella spp., Salmonella Enteritidis, Campylobacter jejuni, C. coli, and Listeria monocytogenes per 100 cm2 or 25 g. Poultry samples were contaminated with Salmonella (36.5%), C. jejuni and C. coli (28.5%), and L. monocytogenes (38.2%). In about 12.3% of the poultry samples, the L. monocytogenes contamination level exceeded 1 CFU per g or cm2. Significant differences in pathogen contamination rates of poultry products were noticed between the poultry products originating from Belgian, French, and U.K. abattoirs. Poultry products derived from broiler chickens running free in pine woods until slaughtering age (12 to 13 weeks) had a significantly (P < 0.05) lower contamination rate of Salmonella than poultry products from enclosed broilers slaughtered at the age of 6 to 8 weeks. A significantly (P < 0.05) lower pathogen contamination rate was noted for Salmonella, C. jejuni, and C. coli for poultry cuts without skin compared to poultry cuts with skin on. An increase in pathogen contamination rate was noticed during cutting and further processing. To diminish C. jejuni, C. coli, Salmonella, and L. monocytogenes contamination rates, hygienic rules of slaughter and meat processing must be rigorously observed. At the moment, zero tolerance for these pathogens is not feasible, and there is a need to establish criteria allowing these pathogens to be present at reasonable levels in the examined poultry samples.     

Potential competitive exclusion bacteria from poultry inhibitory to Campylobacter jejuni and Salmonella

The objective of this study was to isolate from chickens potential competitive exclusion bacteria (CE) that are inhibitory to Campylobacter jejuni or Salmonella, or to both, for subsequent development of a defined CE product for use in poultry. Adult chickens from family farms, commercial farms, and broiler chicken research centers were sampled to identify and select C. jejuni-free donor chickens. A challenge treatment, which included administering perorally 106 CFU C. jejuni per chicken and determining undetectable cecal shedding of campylobacters at 4 weeks, was important for identifying the best CE donor chickens. Screening of bacterial colonies obtained from nine donor chickens by using selective and nonselective media yielded 636 isolates inhibitory to six C. jejuni strains in vitro, with 194 isolates being strongly inhibitory. Of the 194 isolates, 145 were from ceca, and 117 were facultative anaerobic bacteria. One hundred forty-three isolates were inhibitory to six strains of Salmonella (including five different serotypes) in vitro. Of these, 41 were strongly inhibitory to all C. jejuni and Salmonella strains evaluated, and most were Lactobacillus salivarius. A direct overlay method, which involved directly applying soft agar on plates with discrete colonies from mucus scrapings of gastrointestinal tracts, was more effective in isolating CE than was the frequently practiced isolation method of picking and transferring discrete colonies and then overlaying them with soft agar. The best approach for obtaining bacteria highly inhibitory to Salmonella and C. jejuni from chickens was to isolate bacteria from ceca under anaerobic conditions. Free-range chickens from family farms were better donors of potential CE strongly inhibitory to both Salmonella and Campylobacter than were chickens from commercial farms and broiler chicken research centers.     

Molecular epidemiology and antimicrobial susceptibility of Campylobacter jejuni and Campylobacter coli isolates of poultry, swine, and cattle origin collected from slaughterhouses in Hungary

Campylobacter spp. are the most common cause of bacterial enteritis in Hungary, and the aim of this study was to identify the distribution, genotypes, and antimicrobial susceptibility of Campylobacter species in the most important food-producing animals at the time of slaughter during 2008 and 2009. Of 1,110 samples, 266 were identified as Campylobacter coli (23.9%) and 143 as C. jejuni (12.9%) by real-time PCR. Resistance to enrofloxacin-ciprofloxacin and nalidixic acid was significant, especially in C. jejuni (73.3%) and C. coli (77.2%) from broilers. Higher erythromycin (P = 0.043) and tetracycline (P = 1.865e-14) resistance rates were found among C. coli isolates (9.7 and 74.1%, respectively) than among C. jejuni isolates (3.1 and 36.6%, respectively). A total of 47 fla short variable region sequences were identified among 73 selected C. coli and C. jejuni isolates, with 35 fla types detected only once. At the nucleotide level, fla types A66 and A21 were the most common. Using the pulsed-field gel electrophoresis method, 66% of strains exhibited unique profiles after Sma I digestion. Forty-two isolates assigned to 18 Sma I clusters were further typed by Kpn I, and of these, 24 were assigned to 10 Kpn I clusters. For isolates in five Kpn I clusters, epidemiological links were observed. Stable C. jejuni and C. coli clones were detected, indicating that further studies involving broiler and human isolates need to be conducted to elucidate the importance of these stable clones in human infections.     

含内标的多重实时PCR法同时检测空肠弯曲菌和结肠弯曲菌

目的建立含内标的多重实时荧光PCR法同时检测空肠弯曲菌和结肠弯曲菌。方法针对空肠弯曲菌特有hipO基因和结肠弯曲菌特有ceuE基因设计引物探针,设计并优化内标DNA添加量。测试了方法的特异性、灵敏度以及在鸡肉中的检出限。结果内标的最适添加量为10~4copies/PCR。所建立方法对空肠弯曲菌和结肠弯曲菌的灵敏度分别达到4.7copies/PCR和5.23copies/PCR;对115株空肠弯曲菌、49株结肠弯曲菌和42株非目标菌株在3种不同类型的实时荧光PCR仪上的特异性均达到100%;对鸡肉中空肠弯曲菌和结肠弯曲菌的检出限达到10CFU/25g,与传统检测方法一致。采用所建立的方法对50份市售生鲜鸡肉进行检测发现,空肠弯曲菌阳性率为12%(6/50),结肠弯曲菌阳性率为4%(2/50);传统国标检测方法除了1份空肠弯曲菌阳性样品未得到分离确认,其余PCR阳性样品均在平板上分离确认。结论该方法特异性强、灵敏度高、开放性好、含有内标可防止"假阴性",可应用于食品中2种重要致病性弯曲菌的快速同步检测。     

A multiplex polymerase chain reaction for the differentiation of Campylobacter jejuni and Campylobacter coli from a swine processing facility and characterization of isolates by pulsed-field gel electrophoresis and antibiotic resistance profiles

A multiplex polymerase chain reaction (PCR) was developed for the detection and speciation of 60 Campylobacter strains isolated from porcine rectal swabs and from different areas in a pork processing plant. The PCR assay was based on primers specific for the cadF gene of pathogenic Campylobacter species, a specific but undefined gene of Campylobacter jejuni, and the ceuE gene of Campylobacter coli. Further characterization of these isolates was established by pulsed-field gel electrophoresis (PFGE) analyses with the restriction endonuclease SmaI. In addition to molecular discrimination, the antibiotic resistance profiles of the isolates were examined by the Kirby Bauer disc diffusion method with 22 antibiotics. Differentiation of isolates by multiplex PCR identified 86.9% (52 of 60) as C. coli and 13.1% (8 of 60) as C. jejuni. Using the Molecular Analyst software, 60 PFGE types were identified. The percentages of relatedness among C. jejuni strains with PFGE ranged from 25 to 86%, while those among C. coli strains ranged from 34 to 99%. Among the 60 PFGE types, each of 12 C. coli isolates showed > or =90% similarity to one other isolate. The antibiotic resistance profiles of all 60 isolates were distinct. Analyses of antibiotic resistance profiles showed that all isolates were resistant to five or more antibiotics. Twenty-five percent (2 of 8) of C. jejuni isolates and 15% (8 of 52) of C. coli isolates were resistant to at least one of the three fluoroquinolones tested, antibiotics that are commonly used in the treatment of human Campylobacter infections. Three percent (2 of 60) of Campylobacter isolates examined were resistant to all three fluoroquinolones. On the basis of the PFGE and antibiotic resistance profiles, each of the 60 isolates was distinct, suggesting that C. jejuni and C. coli strains originating from diverse sources were present in porcine samples and in the pork processing plant.     

Antimicrobial resistance and molecular subtyping of Campylobacter jejuni and Campylobacter coli from retail meats

Campylobacter isolates (n = 297; 202 C. jejuni and 95 C. coli isolates) recovered from 2,513 retail meat samples (chicken breasts, ground turkey, ground beef, and pork chops) were examined for antimicrobial susceptibility. The isolates were further analyzed for genetic relatedness by pulsed-field gel electrophoresis (PFGE) using SmaI and KpnI restriction enzymes, and a subset of isolates (n = 174) were subtyped by multilocus sequence typing (MLST). The resistance most frequently observed was that to doxycycline (27.6%), followed by ciprofloxacin (13.8%) and erythromycin (6.4%). All isolates were susceptible to gentamicin and meropenem. C. coli showed higher resistance to doxycycline than did C. jejuni (42.1 versus 20.8%) and lower resistance to ciprofloxacin than did C. jejuni (10.5 versus 15.3%). Erythromycin resistance was only observed in C. coli. PFGE using SmaI plus KpnI digestion generated 168 clusters from 297 isolates: 115 from C. jejuni and 53 from C. coli. MLST revealed 44 sequence types (STs) under 10 clonal complexes from 120 C. jejuni and 27 STs under two clonal complexes from 54 C. coli. There was a positive association between PFGE and STs; however, PFGE showed greater discriminatory power than MLST. Subtyping data did not correlate with antimicrobial resistance phenotypes.     

Development of a polymerase chain reaction-based assay for the detection of Alternaria fungal contamination in food products.

Alternaria sp. are important fungal contaminants of vegetable, fruit, and grain products, including Alternaria alternata, a contaminant of tomato products. To date, the Howard method, based on microscopic observation of fungal filaments, has been the standard examination for inspection of tomato products. We report development of a polymerase chain reaction (PCR)-based method for detection of Alternaria DNA. PCR primers were designed to anneal to the internal transcribed regions ITS1 and ITS2 of the 5.8S rRNA gene of Alternaria but not to other microbial or tomato DNA. We demonstrate use of the PCR assay to detect Alternaria DNA in experimentally infested and commercially obtained tomato sauce and tomato powder. Use of the PCR method offers a rapid and sensitive assay for the presence of Alternaria DNA in tomato products. The apparent breakdown of DNA in tomato sauce may limit the utility of the assay to freshly prepared products. The assay for tomato powder is not affected by storage time.     

High pressure inactivation of Escherichia coli, Campylobacter jejuni, and spoilage microbiota on poultry meat

This study evaluated the high pressure inactivation of Campylobacter jejuni, Escherichia coli, and poultry meat spoilage organisms. All treatments were performed in aseptically prepared minced poultry meat. Treatment of 19 strains of C. jejuni at 300 MPa and 30°C revealed a large variation of pressure resistance. The recovery of pressure-induced sublethally injured C. jejuni depended on the availability of iron. The addition of iron content to enumeration media was required for resuscitation of sublethally injured cells. Survival of C. jejuni during storage of refrigerated poultry meat was analyzed in fresh and pressuretreated poultry meat, and in the presence or absence of spoilage microbiota. The presence of spoilage microbiota did not significantly influence the survival of C. jejuni. Pressure treatment at 400 MPa and 40°C reduced cell counts of Brochothrix thermosphacta, Carnobacterium divergens, C. jejuni, and Pseudomonas fluorescens to levels below the detection limit. Cell counts of E. coli AW1.7, however, were reduced by only 3.5 log (CFU/g) and remained stable during subsequent refrigerated storage. The resistance to treatment at 600 MPa and 40°C of E. coli AW1.7 was compared with Salmonella enterica, Shiga toxin-producing E. coli and nonpathogenic E. coli strains, and Staphylococcus spp. Cell counts of all organisms except E. coli AW 1.7 were reduced by more than 6 log CFU/g. Cell counts of E. coli AW1.7 were reduced by 4.5 log CFU/g only. Moreover, the ability of E. coli AW1.7 to resist pressure was comparable to the pressure-resistant mutant E. coli LMM1030. Our results indicate that preservation of fresh meat requires a combination of high pressure with high temperature (40 to 60°C) or other antimicrobial hurdles.     

Campylobacter fetus ssp. jejuni: Recovery Methodology and Isolation from Lamb Carcasses

Campylobacter fetus ssp. jejuni has increasingly been implicated as the causative agent in human cases of gastroenteritis. The first account of C. jejuni isolation from red meats—lamb carcasses—is reported. Recovery of Campylobacter from meat surfaces was tested as follows: selective agar plates consisted of tryptic soy agar, 5% lysed defibrinated horse blood, 10 mg/L vancomycin, 2500 units/L polymyxin B, 5 mg/L trimethoprim lactate, and 15 mg/L cephalothin (Keflin; VPTK: plates). These VPTK plates were incubated at 35°C for 72 hr in a gas atmosphere of 5% O₂, 10% CO₂, and 85% N₂. With these selective elements, recovery from meat surfaces inoculated with a calculated 32 Campylobacter cells per cm² was accomplished in 5 of 5 replicate tests. At levels of 3.2 and 0.3 cells of Campylobacter per cm², recoveries were accomplished in 2 of 5 replicate tests at each inoculum level.     

Reduction of Campylobacter jejuni on poultry by low-temperature treatment

Campylobacter jejuni is a leading cause of acute bacterial gastroenteritis in the United States, with epidemiologic studies identifying poultry as a leading vehicle in human infection. Studies were conducted to determine rates of C. jejuni inactivation on poultry exposed to different cooling and freezing temperatures. A mixture of three strains of C. jejuni originally isolated from poultry was inoculated onto chicken wings at ca. 10(7) CFU/g. The results of the study revealed that the storage of wings at -20 and -30 degrees C for 72 h reduced the population of C. jejuni on wings by 1.3 and 1.8 log10 CFU/g, respectively. The results with regard to long-term freezing for 52 weeks revealed C. jejuni reductions of ca. 4 and 0.5 log10 CFU/g on wings held at -20 and -86 degrees C, respectively. Protocols were developed to superchill wings in Whirl-Pak bags with liquid nitrogen at -80, -120, -160, and -196 degrees C such that the internal portion of each wing quickly reached -3.3 degrees C but did not freeze. The results with regard to the superchilling of wings at different temperatures for 20 to 330 s (the time required for the wings to reach an internal temperature of -3.3 degrees C) revealed C. jejuni reductions of 0.5 log10 CFU/g for wings held at -80 degrees C, 0.8 log10 CFU/g for wings held at -120 degrees C, 0.6 log10 CFU/g for wings held at -160 degrees C, and 2.4 log10 CFU/g for wings held at -196 degrees C. The superchilling of wings to quickly cool meat to -3.3 degrees C (internal temperature) can substantially reduce C. jejuni populations at -196 degrees C when the wings are submerged in liquid nitrogen, but not at -80 to -160 degrees C when the wings are treated with vapor-state liquid nitrogen. The results of this study indicate that freezing conditions, including temperature and holding time, greatly influence the rate of inactivation of C. jejuni on poultry. The conditions used in the poultry industry to superchill poultry to a nonfrozen-state internal temperature are not likely to substantially reduce Campylobacter populations on fresh products.     

One-step polymerase chain reaction-based typing of Arcobacter species

A species-specific PCR assay was developed for the identification of the Arcobacter species, Arcobacter butzleri, Arcobacter cryaerophilus 1A and 1B, and Arcobacter skirrowii. The primers, which amplify the most variable areas of the 23S rRNA gene, were designed to perform species-specific identification by one-step PCR. The DNA sequence of the region from A. cryaerophilus 1B was determined, and the specific pimer for the species was designed. By using one-step PCR containing the mixed primers N.butz, N.c1.A, N.c.1B, and N.ski, species-specific amplifications were detected from the reference strains of A. butzleri, A. cryaerophilus 1A, 1B, and A. skirrowii, respectively. Primers designed in this study were also evaluated on 10 of Japanese field isolates, and all species were identified. This simple one-step PCR assay was found to be a powerful tool for the survey of Arcobacter infection.